scholarly journals Optimization of Conditions for In Vitro Culture of the Microphallid DigeneanGynaecotyla adunca

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Jenna West ◽  
Alexandra Mitchell ◽  
Oscar J. Pung
Keyword(s):  
Egg Production ◽  
Horse Serum ◽  
Calf Serum ◽  
Culture Conditions ◽  
In Vitro Cultivation ◽  
Ilyanassa Obsoleta ◽  
Uca Pugnax ◽  
Newborn Calf Serum ◽  
Chicken Serum

In vitro cultivation of digeneans would aid the development of effective treatments and studies of the biology of the parasites. The goal of this study was to optimize culture conditions for the trematode,Gynaecotyla adunca. Metacercariae of the parasite from fiddler crabs,Uca pugnax, excysted in trypsin, were incubated overnight to permit fertilization, and were cultured in different conditions to find those that resulted in maximum worm longevity and egg production. When cultured in media lacking serum, worms lived longer in Hanks balanced salt solution and Dulbecco’s Modified Eagle medium/F-12 (DME/F-12) than in RPMI-1640 but produced the most eggs in DME/F-12. Worm longevity and egg production increased when worms were grown in DME/F-12 supplemented with 20% chicken, horse, or newborn calf serum but the greatest number of eggs was deposited in cultures containing horse or chicken serum. Horse serum was chosen over chicken serum due to the formation of a precipitate in chicken serum. The optimal concentration of horse serum with respect to egg production ranged from 5 to 20%. Infectivity of eggs deposited by worms in culture was tested by feeding eggs to mud snails,Ilyanassa obsoleta. None of these snails producedG. aduncacercariae.

The in vitro cultivation of Hymenolepis microstoma from cysticercoid to egg-producing adult

1970 ◽  
Vol 48 (5) ◽  
pp. 1135-1137 ◽  
Author(s):  
W. S. Evans
Keyword(s):  
Growth And Development ◽  
Egg Production ◽  
Liver Extract ◽  
Horse Serum ◽  
Body Growth ◽  
In Vitro Cultivation ◽  
General Body ◽  
Hymenolepis Microstoma

Cysticercoids of Hymenolepis microstoma have been cultured in vitro to egg-producing adults in 16 days using Eagle's medium with horse serum plus sheep or hamster liver extract, and ox bile. Bile was essential for egg production but not for general body growth. Worms became gravid when cultured under 0, 5, and 10% oxygen. However, growth and development was greatly inhibited when higher concentrations of oxygen were used.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

Effect of culture conditions on artificial activation of porcine oocytes matured in vitro

1996 ◽  
Vol 8 (8) ◽  
pp. 1153 ◽  
Author(s):  
N Yamauchi ◽  
H Sasada ◽  
S Sugawara ◽  
T Nagai
Keyword(s):  
In Vitro Maturation ◽  
Fetal Calf Serum ◽  
Culture Media ◽  
Calf Serum ◽  
Treated Group ◽  
Culture Conditions ◽  
Culture Period ◽  
Subsequent Response ◽  
Artificial Activation

The effects of culture media used and culture period for in vitro maturation of porcine oocytes on their subsequent response to chemical and electrical activation, were investigated. Activated oocytes were identified by the presence of a pronucleus(ei) or cleavage. Porcine oocytes were cultured for 24, 30, 36, 42 and 48 h in TCM199 with Earle's salts (199) supplemented with 10% fetal calf serum (199-FCS) before electrical stimulation. Although few oocytes were activated after 24 h and 30 h of culture (5.4% and 6.1% respectively), the percentage of activated oocytes increased significantly to 93.2% after 42 h in culture (P < 0.05); however, when the culture period was extended to 48 h, there was a significant decrease to 56.7% (P < 0.05). Oocytes were also cultured in four types of media: (1) 199-FCS; (2) 199 supplemented with 5 mg mL-1 bovine serum albumin (199-BSA); (3) Kreb's-Ringer bicarbonate solution supplemented with 10% FCS (KRB-FCS); and (4) KRB supplemented with BSA (KRB-BSA). After 42 h of culture in each medium, the oocytes were electrically activated. Although rates of maturation of oocytes cultured in the four media were similar (74.0-80.8%), all oocytes except those cultured in 199-FCS failed to be activated. In addition, oocytes were cultured for 36, 42 and 48 h in 199-FCS and then stimulated by treatment with ethanol. Significantly fewer oocytes were activated in the chemically-treated group than in the electrically-treated group. These results indicate that culture conditions used for the culture of porcine oocytes in vitro are important with respect to their subsequent response to artificial activation.

149 EFFECTS OF FSH ADDED IN DEFINED MEDIUM MATURATION ON THE PRE-IMPLANTATION DEVELOPMENT AND EXPRESSION OF Bax, Bcl-2, AND CONNEXIN 43 GENES IN BOVINE IVP BLASTOCYSTS

2010 ◽  
Vol 22 (1) ◽  
pp. 233
Author(s):  
L. V. M. Gulart ◽  
L. Gabriel ◽  
L. P. Salles ◽  
G. R. Gamas ◽  
D. K. Souza ◽  
...  
Keyword(s):  
Internal Control ◽  
Connexin 43 ◽  
Calf Serum ◽  
Defined Medium ◽  
Culture Conditions ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Blastocyst Rate

FSH at low concentrations affect embryo production. In vitro culture conditions also affect embryo production and embryonic expression of genes and alter oocyte competence to produce embryos. The search for better and less variable culture conditions simulating those in vivo has led to the development of several systems of oocyte in vitro maturation culture. To compare the efficiency of the systems of MIV we utilized 4 groups: (1) TCM-199 control; (2) α-minimal essential medium (MEM); 3) α-MEM + 1 ng of FSH; 4) α-MEM+ 10 ng of FSH. The medium of Group 1 is non-defined by the presence of fetal calf serum (10%). Groups 2, 3, and 4 are defined and polyvinyl alcohol (1%) was used as a macromolecule. Porcine FSH (1 IU mg-1) was used at 1 and 10 ng mL-1 and at 100 ng in defined and non-defined medium, respectively. Bovine ovaries were collected at an abbatoir. Oocytes (n = 1718) with homogeneous cytoplasm and with more than 3 layers of granulosa cells were used. Mature oocytes from the 4 treatments (11 replicates of each treatment) were inseminated with frozen-thawed, motile sperm separated by Percoll, using Sperm TALP HEPES medium. Presumptive zygotes with up to 2 or 3 layers of cumulus cells were cultured in 50-mL drops of SOF medium, supplemented with 10% FCS and 1 mg mL-1 BSA under mineral oil in a humid 5% CO2 atmosphere at 38.5°C after. Cleavage rate was evaluated 72 h post-insemination (hpi), and blastocyst rate was evaluated 168-192 hpi. Cleavage and blastocyst rates were calculated on the basis of number of presumptive zygotes. The expression of the following genes (Bax, Bcl-2, and conexin 43) was evaluated in blastocysts by RT-PCR. One-way ANOVA was used to compare blastocyst number. There was no difference in the proportion of embryos with more than 8 blastomeres in all groups tested, indicating that the rate of development during the first 72 hpi was similar for oocytes matured in chemically defined medium and for oocytes matured in medium containing serum. Bax is a pro-apoptotic marker and Bcl-2 an antiapoptotic marker. Connexin 43 (Cx43) may be a marker of embryo competence. Glyceraldehyde 3-phosphate dehydrogenase was used as internal control. The Bax gene was not expressed in any group. The Bcl-2 and Cx43 genes were expressed, mainly in the α-MEM 10. Although no differences were observed in blastocyst rate among the groups (30% to 40%), the strong expression of Bcl-2 and of Cx43 on the group containing 10 ng mL-1 of FSH may indicate that FSH could improve embryo quality under defined conditions. The authors thank FAP-DF, CNPq, FUNPE, FINATEC, CAPES, and Biovitro Tecnologia de Embrioes Ltda, for laboratory assistance and grants, and Frigorifico Ponte Alta, Brasília-DF, for supplying bovine ovaries.

scholarly journals Oviposition by Schistosoma mansoni during in vitro cultivation

1996 ◽  
Vol 38 (6) ◽  
pp. 423-426 ◽  
Author(s):  
Leo Roberto Barth ◽  
Ana Paula Morais Fernandes ◽  
Vanderlei Rodrigues
Keyword(s):  
Schistosoma Mansoni ◽  
Egg Production ◽  
Culture Medium ◽  
Parasite Development ◽  
In Vitro Cultivation ◽  
Progressive Reduction ◽  
Maximum Period ◽  
Medium Time ◽  
Kinetics Of

Observation of Schistosoma mansoni oviposition during in vitro culture of adult worms for a maximum period of 10 days showed three well distinct phases in the kinetics of oviposition: an initial phase with low egg production, a period of maximum oviposition and finally a progressive reduction in the number of eggs during the late phases of culture. The kinetics of oviposition and the number of eggs laid by the parasites are influenced by the number of worm pairs per amount of RPMI 1640 medium, time of parasite development in the vertebrate host and type of serum utilized in the culture medium.

Oesophagostomum radiatum: inhibition of in vitro development by newborn calf serum

1990 ◽  
Vol 64 (1) ◽  
pp. 9-14
Author(s):  
I. J. East ◽  
C. J. Fitzgerald
Keyword(s):  
Inhibitory Action ◽  
Natural Infection ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Newborn Calf Serum ◽  
In Vitro Development ◽  
Specific Antibodies ◽  
Serum Inhibition ◽  
Vitro Development

ABSTRACTOesophagostomum radiatum developed to fourth stage larvae after 14 days in in vitro culture. However, development was totally inhibited if the standard 50% foetal calf serum in the medium was replaced by newborn calf serum. Inhibition did not occur with serum from cattle immune to O. radiatum through natural infection or experimental vaccination irrespective of the titre of specific antibodies to O. radiatum in each serum. The inhibitory action of NCS could be abolished by heat treatment at 56°C for 1 h but not by dialysis or repeated freeze-thawing. The inhibition was not consistent with observed differences in the activity of 19 enzymes in the various sera or the absence of various thiol-containing stimulants of worm development.

Preliminary experiments on the in vitro cultivation of an amphistome Orthocoelium scoliocoelium (Trematoda: Digenea)

1982 ◽  
Vol 56 (3) ◽  
pp. 169-176 ◽  
Author(s):  
A. N. Sharma ◽  
P. N. Sharma
Keyword(s):  
Amino Acids ◽  
Egg Production ◽  
Rumen Fluid ◽  
Inorganic Salts ◽  
In Vitro Cultivation ◽  
Short Term ◽  
Physiological Alterations

ABSTRACTA medium containing inorganic salts, vitamins and amino-acids is described for the in vitro cultivation of the amphistome, Orthocoelium scoliocoelium, from the rumen of buffalo. Based on the properties of rumen fluid, this medium promoted and sustained normal egg production, at 37°C temperature and pH 7·4 to 7·8 for 15 days. In contrast to other media used, no patho-physiological alterations except abnormal cytoplasmic lipid, were detected. Since the parasites produced normal eggs, it is concluded that this medium is suitable for short-term cultivation of the parasites.

Bovine in vitro oocyte maturation as a model for manipulation of the γ-glutamyl cycle and intraoocyte glutathione

2008 ◽  
Vol 20 (5) ◽  
pp. 579 ◽  
Author(s):  
E. C. Curnow ◽  
J. Ryan ◽  
D. Saunders ◽  
E. S. Hayes
Keyword(s):  
Oocyte Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Buthionine Sulfoximine ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Maturation Medium

Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels.

Effect of Phytohemagglutinin on Mouse Peripheral Lymphocytes.

1967 ◽  
Vol 53 (6) ◽  
pp. 565-574 ◽  
Author(s):  
Giuseppe Tridente ◽  
Gian Maria Cappuzzo ◽  
Luciano Fiore-Donati
Keyword(s):  
Inbred Mice ◽  
Calf Serum ◽  
Culture Conditions ◽  
Culture Technique ◽  
Mouse Strains ◽  
Thymidine Uptake ◽  
Peripheral Lymphocytes ◽  
Cellular Factors ◽  
Pha Stimulation

Using a short-term culture technique it has been possible to cultivate mouse peripheral lymphocytes from various strains for as long as 96 hours. With this technique a study was made to determine whether mouse peripheral lymphocytes could be stimulated by PHA « in vitro ». Swiss (non-inbred) and CBA (inbred) mice of both sexes, 3–12 months old, were used throughout this study. Blood was obtained from the retro-orbital sinus of ether-anesthetized animals, collected in heparinized tubes and pooled according to sex and strain. Leukocyte separation was effected by sedimentation in Plasmagel for 15'. The supernatant was transferred to an equal volume of Hanks Balanced Salt Solution (HBSS) and spun down for 20’ at 250–300 × g. The resulting cell sediment was washed twice with HBSS and resuspended in 2 ml Trowell T8 medium which contained 30 - 40 % calf serum which had been pre-heated for 30’ in a 56 °C water bath. After a differential cell count, the cell suspension was further diluted to achieve 106 lymphocytes/ml. Red blood cell contamination corresponded to 30 rbc/1 wbc with granulocyte population varying 10–40 %. 3 × 106 lymphocytes were placed in glass culture tubes prepared in advance by adding 2 ml of 1.5 % agar in HBSS and allowing it to solidify in the bottom of upright tubes. 0.1 ml PHA-M was added to each tube and the cultures were kept upright in a controlled gas phase incubator for 24–96 hours at 37 °C. At the end of the incubation period, the cultures were transferred to centrifuge tubes and spun down for 10’ as above and smears were made directly from the dense sediment after the supernatant was discarded. These smears were fixed, stained and read for the presence of transformed cells scoring at least 1000 cells/culture. Some cultures received 3 μg colchicine 2–6 hours before harvesting and chromosome preparations were made. Mitotic indexes were estimated by scoring 1000 cells/culture. Swiss cultures were also employed for radioautography by adding 3H-thymidine 6 hours before harvesting. Cells were then washed once in HBSS, subjected to short hypotonic treatment, rapidly fixed, pipetted on to slides and allowed to dry. Slides were coated with Kodak NTB2 emulsion, exposed for 9 days, processed and stained through the film and read for labelled cells scoring 500 cells/slide. Microscopic examination revealed a prompt Mastoid transformation of the small lymphocytes which presented a deeply basophilic cytoplasm surrounding a large nucleus with reticular chromatin and one or more prominent nucleoli. Quantitation of the blastoid response at 24-hour intervals throughout the 96 hours of culture disclosed significant blast formation within the first 24 hours for both mouse strains. Differences in transformation between the 2 strains were statistically significant at 24 and 48 hours. 3H-thymidine uptake paralleled blastogenesis by increasing progressively. Differences in uptake between PHA cultures and corresponding controls were found to be statistically significant for all except the 96 - hour cultures. The results obtained suggest that mouse peripheral lymphocytes will respond to PHA stimulation provided culture conditions are adequate. There was clear evidence of stimulation by PHA in both morphology and radioautography studies. However, the day-to-day variation in values of transformation and 3H-thymidine uptake necessitate further studies to improve culture conditions. Higher variability in Swiss (non-inbred) cultures indicates that cellular factors may also influence response « in vitro ». It is concluded that mouse peripheral lymphocytes react « in vitro » to PHA in much the same way as human and other mammalian lymphocytes.

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